Loss of proteostasis is a hallmark of aging. It has been proposed that this loss is due to a decline in many biological processes used by cells to regulate and maintain their proteins, from synthesis to degradation. Loss of proteostasis results in the accumulation of damaged proteins and aggregates, leading to impaired cellular function and cell death, as well as a range of age-related diseases, like Alzheimer's and Parkinson's disease. Cells that are particular sensitive to loss of proteostasis are the stem cells (SCs) found in regenerative tissues, such as the intestine. These somatic SCs are needed to regenerate tissues after normal cell attrition or in response to stress. Somatic SCs have been proposed to have unique mechanisms for maintaining their proteostatic capacity. However, these mechanisms are not completely understood. This proposal aims at understanding how somatic SCs maintain their proteostasis, why this is lost during the aging process, and at exploring ways of improving proteostasis in old tissues. To answer these questions, the applicant proposes studies using the fruit fly Drosophila melanogaster as a model organism. Through work by the mentor and others, the fly posterior midgut has emerged as a powerful genetically amenable system to study intestinal SC (ISC) function in the context of tissue regeneration and aging. In preliminary studies, the applicant has extended this work to develop an experimental paradigm to establish the effectiveness of protein aggregate clearance in ISCs under different environmental and genetic conditions in vivo. This work has led to the observation that ISCs react to protein aggregates by undergoing temporary cell cycle arrest while clearing aggregates, and that this arrest, as well as the ability to clear aggregates, declines with age. Based on these observations and on the current literature, the applicant hypothesizes that ISCs activate a 'proteostasis checkpoint' regulated by the transcription factor Nrf2/CncC whenever proteostasis is compromised. Nrf2/CncC is a master regulator of the antioxidant response that regulates ISC proliferation and controls the expression of genes encoding one of the degradation machineries, the proteasome. To test this hypothesis, the applicant proposes genetic studies in ISCs to: (i) assess the role of Nrf2/CncC in the proteostasis checkpoint and in aggregate clearance, (ii) explore the mechanism of aggregate clearance, and (iii) test if promoting proteostasis in old ISCs is sufficient to improve tissue homeostasis and survival. The proposed studies will characterize specific mechanisms by which somatic stem cells maintain protein homeostasis throughout life, and will gain insight into whether and how these mechanisms influence tissue homeostasis and survival. Based on the evolutionary conservation of many of the regulatory mechanisms studied in this research proposal, it can be anticipated that the findings obtained by this work will have important implications for human disease and aging.